U.S. patent application number 14/659414 was filed with the patent office on 2015-07-09 for fluoride processing method.
This patent application is currently assigned to GE HEALTHCARE LIMITED. The applicant listed for this patent is GE Healthcare Limited. Invention is credited to Rajiv Bhalla, Alexander Jackson.
Application Number | 20150191477 14/659414 |
Document ID | / |
Family ID | 40612964 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150191477 |
Kind Code |
A1 |
Jackson; Alexander ; et
al. |
July 9, 2015 |
Fluoride Processing Method
Abstract
The invention relates to methods for processing
[.sup.18F]-fluoride target water using a solid-support bound
Cryptand of formula (I) ##STR00001## and to apparatus for
performing such methods. The resultant [.sup.18F]-fluoride is
useful for preparation of radiopharmaceuticals by nucleophilic
fluorination, specifically for use in Positron Emission Tomography
(PET).
Inventors: |
Jackson; Alexander; (Little
Chalfont, GB) ; Bhalla; Rajiv; (Little Chalfont,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GE Healthcare Limited |
Little Chalfont |
|
GB |
|
|
Assignee: |
GE HEALTHCARE LIMITED
Little Chalfont
GB
|
Family ID: |
40612964 |
Appl. No.: |
14/659414 |
Filed: |
March 16, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12810893 |
Jun 28, 2010 |
8980221 |
|
|
PCT/EP2008/068155 |
Dec 22, 2008 |
|
|
|
14659414 |
|
|
|
|
61018691 |
Jan 3, 2008 |
|
|
|
Current U.S.
Class: |
422/159 ;
540/472 |
Current CPC
Class: |
C01B 9/08 20130101; C07B
59/002 20130101; C07D 487/08 20130101; B01J 2219/00781 20130101;
B01J 19/0093 20130101 |
International
Class: |
C07D 487/08 20060101
C07D487/08; B01J 19/00 20060101 B01J019/00 |
Claims
1. A compound of formula (I): ##STR00033## wherein the Cryptand is
of formula (C): ##STR00034## wherein: R1 and R2 are independently
selected from ##STR00035## ##STR00036## and R3, R4, and R5 are
independently selected from: ##STR00037##
2. A compound of formula (I) according to claim 1 wherein the
Cryptand is selected from ##STR00038##
3. An apparatus for preparation of an [.sup.18F]fluoride solution
which comprises: (i) a vessel containing a solid-support bound
Cryptand of formula (I) as defined in any one of claims 1 to 3;
(ii) means for contacting a solution of [.sup.18F]fluoride in water
with said solid-support bound Cryptand of formula (I) so as to form
a Cryptand-[.sup.18F]fluoride complex of formula (II) as defined in
any one of claims 1 to 3; (iii) means for removal of excess water
from the Cryptand-[.sup.18F]fluoride complex of formula (II); (iv)
means for washing the Cryptand-[.sup.18F]fluoride complex of
formula (II) with a solution of base, suitably a base having a pKa
of at least 9, so as to release the [.sup.18F]fluoride into
solution.
4. Apparatus according to claim 3 which is a microfabricated
device.
Description
[0001] The present invention relates to methods for processing
[.sup.18F]-fluoride target water, and to apparatus for performing
such methods. The resultant [.sup.18]-fluoride is useful for
preparation of radiopharmaceuticals by nucleophilic fluorination,
specifically for use in Positron Emission Tomography (PET).
[0002] Fluorine-18 is obtained by a variety of nuclear reactions
from both particle accelerators and nuclear reactors, and can be
produced at specific activities approaching
1.71.times.10.sup.9Ci/mmol. The half-life of fluorine-18 is 109.7
minutes, relatively long in comparison with other commonly used
radioisotopes but still imposing time constraints on processes for
preparing .sup.18F-labelled radiopharmaceuticals.
[0003] Most fluorine-18 is produced by irradiation of an
[.sup.18O]oxygen gas target by the nuclear reaction
.sup.18O(p,n).sup.18F, and isolated as [.sup.18F]fluoride ion in
aqueous solution. In aqueous form, [.sup.18F]fluoride can be
relatively unreactive, and so certain steps are routinely performed
to provide a reactive nucleophilic [.sup.18F]fluoride reagent.
[0004] Following irradiation, a positively charged counterion is
added, most commonly potassium complexed by a cryptand such as
Kryptofix 222 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo [8,8,8]
hexacosan), or alternatively, cesium, rubidium, or a
tetralkylammonium salt. This is commonly achieved by passing the
[.sup.18F]fluoride target water (typically in volumes of 1 to 5 mL)
through an anion exchange resin and eluting with an aqueous organic
solution (typically in a volume of 0.3 to 1 mL) of the counterion,
for example, with a potassium carbonate/Kryptofix solution in
water/acetonitrile. Secondly, the solution is dried, commonly by
azeotroping in the presence of a low-boiling solvent such as
acetonitrile. Automated radiosynthesis apparatus routinely include
such a drying step, typically lasting 9 minutes in the case of
[.sup.18F]FDG synthesis on Tracerlab MX (GE Healthcare). The
compound to be labelled (dissolved in an organic solvent suitable
for performing the subsequent radiosynthesis, usually an aprotic
solvent such as acetonitrile, dimethylsulphoxide or
dimethylformamide) is then added to the dried residue of
[.sup.18F]fluoride and counterion.
[0005] However, there still exists a need for efficient
[.sup.18F]fluoride processing methodologies which allow rapid,
efficient trapping and elution of [.sup.18F]fluoride from target
water. Additionally, there is a need for such methodologies which
are amenable to automation to facilitate improved preparation of
radiopharmaceuticals in the clinical setting.
[0006] Accordingly, the present invention provides a method for
preparing an [.sup.18F]fluoride solution which comprises:
(i) contacting a solution of [.sup.18F]fluoride in water with a
solid-support bound Cryptand of formula (I)
##STR00002##
at a pH of less than 5 so as to form a Cryptand-[.sup.18F]fluoride
complex of formula (II):
##STR00003##
(ii) removal of excess water from the Cryptand-[.sup.18F]fluoride
complex of formula (II); (iii) washing the
Cryptand-[.sup.18F]fluoride complex of formula (II) with a solution
of base, suitably a base having a pKa of at least 9, so as to
release the [.sup.18F]fluoride into solution.
[0007] In formula (I), the Solid Support may be any suitable
solid-phase support which is insoluble in any solvents to be used
in the method but to which the Linker and/or Cryptand may be
covalently bound. Examples of suitable Solid Support include
polymers such as polystyrene (which may be block grafted, for
example with polyethylene glycol), polyacrylamide, or
polypropylene, or glass or silicon coated with such a polymer. The
Solid Support may take the form of small discrete particles such as
beads or pins, or as coatings on a particle, for example, of glass
or silicon, or a coating on the inner surface of a cartridge or
microfabricated device.
[0008] In formula (I), the Linker is a C.sub.1-50 hydrocarbyl group
optionally further including 1 to 10 heteroatoms such as oxygen or
nitrogen. Suitable Linker groups include alkyl, alkenyl, alkynyl
chains, aromatic, polyaromatic, and heteroaromatic rings (for
example, triazoles), and polymers comprising ethyleneglycol, amino
acid, or carbohydrate subunits any of which may be optionally
substituted for example with one or more ether, thiooether,
sulphonamide, or amide functionality.
[0009] The compounds of formula (I) may be pre-conditioned by
treatment with an acid solution to form a protonated derivative, or
may be non-conditioned.
[0010] As used herein, the term "Cryptand" means a bi- or
poly-cyclic multidentate ligand for the fluoride anion. Suitable
Cryptands for binding anions such as fluoride have been reviewed in
J. W. Steed, J. L. Atwood in Supramolecular Chemistry (Wiley, New
York, 2000), pp 198-249; Supramolecular Chemistry of Anions, Eds. A
Bianchi, K Bowmann-James, E. Garcia-Espana (Wiley-VCH, New York,
1997), and P. D. Beer, P. A. Gale, Angew.Chem. 2001, 113, 502;
Angew. Chem. Int. Ed. 2001, 40, 486.
[0011] Suitable Cryptands used herein include those of formula
(C):
##STR00004##
wherein: R1 and R2 are independently selected from
##STR00005## ##STR00006##
and R3, R4, and R5 are independently selected from:
##STR00007##
Preferred Cryptands useful in the invention may be selected
from:
##STR00008##
or may be chosen to have desirable properties such as a high
binding constant for fluoride, high stability of the fluoride bound
complex and high fluoride selectivity over other anions.
[0012] In the compounds of formula (I), the Cryptand is attached to
a Linker group. The point of attachment may be a nitrogen or carbon
atom in the Cryptand. Thus the point of attachment to the Linker
"L" may be in group R1 or R2:
##STR00009##
or in R3, R4, or R5:
##STR00010## ##STR00011##
[0014] The method of the invention may be performed by contacting
the solid-support bound Cryptand of formula (I) with the solution
of [.sup.18F]fluoride in water in a container and then separating
the resulting solid-phase Cryptand-[.sup.18F]fluoride complex of
formula (II) by filtration. Alternatively, and particularly
suitably when the solid-support bound Cryptand of formula (I) is
used within an automated apparatus, the solid-support bound
Cryptand of formula (I) may be contained in a vessel either as
discrete particles or as a coating through which the solution of
[.sup.18F]fluoride in water is passed. The solution of
[.sup.18F]fluoride in water may be passed through the vessel
containing solid-support bound Cryptand of formula (I) as a
continuous flow, for example at a flow rate of from 0.1 ml/min to
100 ml/min, or in batches, so as to permit sufficient residence
time on the solid-phase for the fluoride complexation to occur. As
would be understood by the person skilled in the art, the
solid-support bound Cryptand of formula (I) may be held in any
suitable vessel such as a plastic or metal column, cartridge, or
syringe barrel. The fluoride complexation is conveniently performed
at ambient temperature, but use of non-extreme elevated temperature
(for example up to 120.degree. C., but preferably up to 80.degree.
C.) can increase efficiency of the fluoride complexation.
[0015] Step (iii) of the process ie. washing the
Cryptand-[.sup.18F]fluoride complex of formula (II) with a solution
of base, suitably a base having a pKa of at least 9, so as to
release the [.sup.18F]fluoride into solution is suitably effected
in a similar manner to steps described above, the solid-support
facilitating separation of the [.sup.18F]fluoride in solution. The
base is suitably selected from a potassium salt (such as potassium
carbonate, potassium bicarbonate, or potassium sulphate) optionally
in the presence of a phase transfer catalyst such as Kryptofix; a
tetraalkylammonium salt (such as tetraalkylammonium carbonate,
tetralkylammonium bicarbonate, or tetraalkylammonium sulphate); a
phosphonium salt (such as phosphonium carbonate, phosphonium
bicarbonate, or phosphonium sulphate); a cesium salt (such as
cesium carbonate, cesium bicarbonate, or cesium sulphate); and an
imidazolium salt (such as imidazolium carbonate, imidazolium
bicarbonate, or imidazolium sulphate) and is provided in a solution
comprising organic solvent (suitably selected from acetonitrile,
dimethylformamide, dimethylsulfoxide, tetrahydrofuran, dioxan,
1,2-dimethoxyethane, sulfolane or N-methylpyrrolidinone or a
mixture of any thereof), water, or an organic solvent containing
water. Suitably, the solution is formed in a dry organic solvent
(i.e. containing less than 1000 ppm water), or an organic solvent
containing water at a level which is tolerated in the subsequent
radiofluoridation reaction, for example 1000 ppm to 50,000 ppm
water, preferably 1000 to 15,000 ppm, more preferably 2000 ppm to
7000 ppm, suitably 2500 ppm to 5000 ppm, as is taught in WO
2006/054098. In this way, a further drying step before
radiofluoridation may be avoided.
[0016] In one aspect of the invention, step (iii) is performed
using a small volume of base solution, such as 400 .mu.l or less,
preferably 50 .mu.l or less, and more preferably 1 to 10 .mu.l. The
[.sup.18F]fluoride solution is then obtained in highly concentrated
form which is advantageous as the volume of water present is
correspondingly low which means the customary step of drying the
[.sup.18F]fluoride solution before performing a radiofluoridation
reaction can be shorter or avoided altogether. Also, this aspect of
the invention makes the method amenable to automation, and
particularly in a smaller reaction vessel such as a miniaturised
device.
[0017] An [.sup.18F]fluoride solution produced by the method of the
invention may then be used in radiosynthetic processes, to perform
nucleophilic [.sup.18F]fluoridation of a Vector.
[0018] As used herein, the term "Vector" means a biomolecule
suitable for radiolabelling to form a radiopharmaceutical, such as
a peptide, protein, hormone, polysaccaride, oligonucleotide,
antibody fragment, cell, bacterium, virus, or small drug-like
molecule.
[0019] The reaction of a Vector with an [.sup.18F]-fluoride
solution produced by the method of the invention may be effected at
an elevated temperature, for example up to 200.degree. C. or at
non-extreme temperature, such as 10.degree. C. to 50.degree. C.,
and most preferably at ambient temperature. The temperature and
other conditions for radiofluoridation being selected according to
the exact reaction being performed, nature of reaction vessel,
solvents etc as would be apparent to a person skilled in the
art.
[0020] Following [.sup.18F]-fluoridation, a purification step may
be required which may comprise, for example, removal of excess
[.sup.18F]-fluoride, removal of solvent, and/or separation from
unreacted Vector. Excess [.sup.18F]-fluoride may be removed by
conventional techniques such as ion-exchange chromatography (for
example using BIO-RAD AG 1-X8 or Waters QMA) or solid-phase
extraction (for example, using alumina). Excess solvents may be
removed by conventional techniques such as evaporation at elevated
temperature in vacuo or by passing a stream of inert gas (for
example, nitrogen or argon) over the solution. Alternatively, the
[.sup.18F]-fluoridated Vector may be trapped on a solid-phase, for
example a cartridge of reverse-phase absorbant for example a
C.sub.5-18 derivatized silica, whilst the unwanted excess reagents
and by-products are eluted, and then the [.sup.18F]-fluoridated
Vector may be eluted from the solid-phase in purified form.
[0021] Selection and synthesis of a Solid Support and/or Linker in
a compound of formula (I) may be effected by conventional
techniques of solid phase chemistry, for example as described in
Florencio Zaragoza Dorwald "Organic Synthesis on Solid Phase;
Supports, Linker, Reactions", Wiley-VCH (2000).
[0022] Compounds of formula (I) may be prepared by reacting a
compound of formula (III):
##STR00012##
with a compound of formula (IV):
##STR00013##
wherein the Solid Support and Cryptand are as defined above,
Linker' is a portion of the Linker as defined above, and R.sup.III
and R.sup.IV are reactive groups capable of covalent bonding to
each other so as to complete formation of the Linker. Suitably, one
of R.sup.III and R.sup.IV is an amine and the other is a carboxylic
acid or an activated carboxylic ester, isocyanate or isothiocyanate
such that the compounds of formulae (III) and (IV) may be joined by
simple amide forming reaction. Suitable activated carboxylic esters
include the N-hydroxysuccinimidyl and N-hydroxysulfosuccinimidyl
esters:
##STR00014##
[0023] Alternatively one of R.sup.III and R.sup.IV may be a thiol
and the other a group reactive towards a thiol, such as a maleimide
or an .alpha.-halocarbonyl.
[0024] As would be apparent to the person skilled in the art, it
may also be desirable for the Cryptand in the Compound of formula
(III) to have protection groups on any exposed functional groups
e.g. amino groups to prevent or reduce side-reactions during
conversion to a Compound of formula (I). In these cases the
protection group will be chosen from those commonly used for the
functional group in question e.g tert-butylcarbamate for an amine.
Other suitable protecting groups may be found in Protecting Groups
in Organic Synthesis, Theodora W. Greene and Peter G. M. Wuts,
published by John Wiley & Sons Inc. which further describes
methods for incorporating and removing such protecting groups.
[0025] Certain compounds of formula (I) may be prepared by reacting
a compound of formula (III) wherein R.sup.III is either an amino or
carboxylic acid group with a compound of formula (IV) wherein
R.sup.IV is either a carboxylic acid or amine group respectively.
In these cases a compound of formula (III) may be coupled with a
compound of formula (IV) optionally using in situ activating agents
such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) or
N-Rdimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene-
]-N-methylmethanamonium hexafluorophosphate N-oxide (HATU).
Standard conditions will be used e.g. dimethylformamide (DMF)
solution and a base e.g. triethylamine or diisopropylethylamine.
Alternatively where R.sup.IV in the compound of formula (IV) is a
thiol group, this may be reacted with a compound (III) in which
R.sup.III is a thiol reactive group such as a maleimide or an
.alpha.-halocarbonyl. This reaction may be performed in a pH
buffered solution or an organic solvent. The product compound
having the formula (II) might be purified by preparative high
performance liquid chromatography.
[0026] The Cryptands may be synthesised as described in
US20040267009 A1, Bernard Dietrich, Jean-Marie Lehn, Jean Guilhem
and Claudine Pascard, Tetrehedron Letters, 1989, Vol. 30, No. 31,
pp 4125-4128, Paul H. Smith et al, J. Org. Chem., 1993, 58,
7939-7941, Jonathan W. Steed et al, 2004, Journal of the American
Chemical Society, 126, 12395-12402, Bing-guang Zhang et al, Chem.
Comm., 2004, 2206-2207.
[0027] The synthesis of a Compound of formula (III) may be achieved
as described in the above references for the underivatized
Cryptands with modifications to the starting materials or by
subsequent chemistry, for example, by alkylation of a secondary
amine group of the Cryptand as illustrated in the Examples below.
Compounds of formula (III) may also be prepared as shown in Schemes
2 to 5 in which L and R''' are as defined above for the Compound of
formula (III).
##STR00015##
##STR00016##
##STR00017##
##STR00018##
[0028] Certain solid-support bound cryptands are novel. Therefore,
as a further aspect of the invention, there is provided a compound
of formula (I):
##STR00019##
wherein the Solid Support and Linker are as defined above, and the
Cryptand is of formula (C):
##STR00020##
wherein: R1 and R2 are independently selected from
##STR00021## ##STR00022##
and R3, R4, and R5 are independently selected from:
##STR00023##
[0029] More preferably in this aspect of the invention, the
Cryptand is selected from:
##STR00024##
[0030] Preferred compounds of formula (III) used to prepare the
compounds of formula (I) include:
##STR00025## ##STR00026##
wherein L is a Linker' as defined above, and R.sup.III is a
reactive group as defined above, and is preferably selected from
amine, carboxylic acid, activated carboxylic ester, isocyanate,
isothiocyanate, thiol, maleimide, or .alpha.-halocarbonyl.
[0031] More preferred compounds of formula (III) used to prepare
the compounds of formula (I) include:
##STR00027##
wherein L is a Linker' as defined above, and R.sup.III is a
reactive group as defined above, and is preferably selected from
amine, carboxylic acid, activated carboxylic ester, isocyanate,
isothiocyanate, thiol, maleimide, or .alpha.-halocarbonyl.
[0032] According to a further aspect of the invention there is
provided an apparatus for preparation of an [.sup.18F]fluoride
solution by a method as described above. Therefore, there is
provided an apparatus for preparation of an [.sup.18F]fluoride
solution which comprises:
(i) a vessel containing a solid-support bound Cryptand of formula
(I) as defined above; (ii) means for contacting a solution of
[.sup.18F]fluoride in water with said solid-support bound Cryptand
of formula (I) so as to form a Cryptand-[.sup.18F]fluoride complex
of formula (II) as defined above; (iii) means for removal of excess
water from the Cryptand-[.sup.18F]fluoride complex of formula (II);
(iv) means for washing the Cryptand-[.sup.18F]fluoride complex of
formula (II) with a solution of base, suitably a base having a pKa
of at least 9, so as to release the [.sup.18F]fluoride into
solution.
[0033] In a further embodiment, the apparatus forms part of, or is
in fluid communication with, an automated radiosynthesis apparatus
such that following preparation of the [.sup.18F]fluoride solution
by a method of the invention, the [.sup.18F]fluoride solution is
used in an [.sup.18F]fluoridation reaction. In one embodiment, the
apparatus is a microfabricated device--either dedicated to
preparation of an [.sup.18F]fluoride solution or further
incorporating means to effect a radiofluoridation reaction.
[0034] In use of the apparatus, the solid-support bound Cryptand of
formula (I), would be contacted with a solution of
[.sup.18F]fluoride in water using methods described above to form
the corresponding compound of formula (II) and then washed with a
solution of base, suitably a base having a pKa of at least 9 so as
to release the [.sup.18F]fluoride into solution.
[0035] Reviews of methods for construction of microfabricated
devices and their application inter alia in synthetic chemistry,
may be found in DeWitt, (1999) "Microreactors for Chemical
Synthesis", Current Opinion in Chemical Biology, 3:350-6; Haswell,
Middleton et al (2001) "The Application of Microreactors to
Synthetic Chemistry", Chemical
[0036] Communications: 391-8; Haswell and Skelton (2000) "Chemical
and Biochemical Microreactors", Trends in Analytical Chemistry
19(6), 389-395; and Jensen (2001) "Microreaction Engineering--Is
Small Better?" Chemical Engineering Science, 56:293-303.
[0037] Suitable microfabricated devices for performing the methods
of the invention have a contained network of microchannels or
capillaries having an internal diameter of typically 10-300 .mu.m,
more typically 50-300 .mu.m. The network of microchannels or
capillaries may be etched or otherwise machined on the surface of a
substrate, suitably made of glass or silicon. Alternatively, the
microchannels may be created using polymers (for example PEEK
plastic, cycloolefin copolymer, polydimethylsiloxane, SU8 (an epoxy
based photoresist), epoxy resin, or polymethylmethacrylate) which
may be poured over a master (usually glass), allowed to cure and
then peeled off, or are fabricated by injection moulding, hot
embossing, casting, lithography, or machining.
[0038] The microchannels or capillaries may be sealed through
bonding of a cover plate, suitably made from a metal (for example,
gold or silver) or, more commonly, glass, creating a contained
network capable of manipulating picolitre volumes of liquid or gas.
The sealing method used depends on the materials selected and may
be selected from thermal bonding (for glass chips), anodic bonding
(for silicon chips), and for polymer chips the sealing method may
be selected from clamping, gluing, application of heat and
pressure, and natural adhesion. Flow capacity could be increased
further, for example, by stacking multiple devices. These devices
are designed to be used either with micro syringe pumps (available
from Kloehen Limited, Las Vegas, USA) or under electroosmotic flow
using fused silica capillaries for interfacing with reagents and
analytical systems (such as ultraviolet (UV), capillary
electrophoresis (CE), capillary electrochromatography (CEC),
electrochemical, refractive index, and radioactivity
detectors).
[0039] Where the vessel is a microchannel in a microfabricated
device, it may be coated with a solid-support bound Cryptand of
formula (I) by conventional methods, for example analogous to those
described in WO2005/061110. Surface modification of poluethylene is
reviewed in the book Advances in Polymer Science (Springer
Berlin/Heidelberg ISSN 0065-3195 (Print) 1436-5030 (Online) Volume
169 DOI 10.1007/b13502 Copyright 2004 ISBN 978-3-540-40769-0 DOI
10.1007/b13524 Pages 231-294). Many of the techniques described
will apply to other plastic materials. Where the microfabricated
device is constructed from poly(methyl metacrylate) (PMMA), the
surface of the PMMA may be amine functionalized as described in
Anal. Chem., 72 (21), 5331-5337, 2000. PMMA devices may also be
functionalized as sulfhyril group as described in United States
patent application 20050101006. Photografting allows the
introduction of surface functional groups to a range of polymeric
materials such as polycarbonates, PMMA, polydimethylsiloxane and
polyolefins as described in Rohr, T., Ogletree, F. D., Svec, F.,
Frechet, J. M., "Surface Functionalization of Thermoplastic
Polymers for the Fabrication of Microfluidic Devices by
Photoinitiated Grafting," Adv. Funct. Mater. 2003, 13, 264-70. In
addition, the reactive surface area may be increased by using
chemically grafted three dimentional monoliths which can be
included in a microfabricated device as described in Rohr, T.,
Ogletree, F. D., Svec, F., Frechet, J. M., "Photografting and the
Control of Surface Chemistry in Three-Dimensional Porous Polymer
Monoliths," Macromolecules 2003, 36, 1677-84 and Stachowiak, T. B.,
Rohr, T., Hilder, E. F., Peterson, D. S., Yi, M., Svec, F.,
Frechet, J. M., "Fabrication of Porous Polymer Monoliths Covalently
Attached to the Walls of Channels in Plastic Microdevices,"
Electrophoresis 2003, 24, 3689-93. Furthermore, the microfabricated
device may also cantain dual functionality where for example, in
addition to the covalently bound cryptand, there may be an
additional reagent e.g. solid supported substrate or chemical
scavenger. Dual function devices are described in Peterson, D. S.,
Rohr, T., Svec, F., Frechet, J. M., "Dual-Function Microanalytical
Device by In Situ Photolithographic Grafting of Porous Polymer
Monolith: Integrating Solid-Phase Extraction and Enzymatic
Digestion for Peptide Mass Mapping," Anal. Chem. 2003, 75,
5328-35.
[0040] The invention is illustrated by way of the following
examples, in which these abbreviations are used:
Et.sub.3N: triethylamine R.T.: room temperature MeOH: methanol (t)
BOC: (tertiary) butoxycarbonyl L: litre mL: millilitre hr(s):
hour(s) THF: tetrahydrofuran HPLC: high performance liquid
chromatography DCM: dichloromethane LCMS: liquid chromatography
mass spectrometry NMR: nuclear magnetic resonance TFA:
trifluoroacetic acid
MBq: Mega Bequerel
[0041] RCP: radiochemical purity
EXAMPLES
Example 1
Synthesis of Compound 4
##STR00028##
[0042] Example 1(i)
Synthesis of Compound 1
[0043] A 1 L 3-neck round-bottom flask equipped with a mechanical
stirrer was charged with 16.7 mL of 98% tripropylamine and 0.33 L
of 99% isopropanol, and cooled to -78.degree. C. in a dry
ice-isopropanol bath. To this mixture, solutions of 15.0 g 40%
aqueous glyoxal (0.103 mole), diluted to 83 mL with isopropanol,
and 10.0 g (0.0.683 moles) of 96% tris-(2-aminoethyl)amine (tren),
diluted to 83 mL, were simultaneously added over a period of 2 hrs
with vigorous stirring. (Initial concentration of glyoxal=1.24 M;
Initial concentration of tren=0.82 M). Then the reaction mixture
was allowed to warm up overnight and briefly warmed up to
60.degree. C. to ensure that the formation of compound 2 was
complete. It was cooled to room temperature while nitrogen gas was
blown over its surface. The solvent was removed under vacuum and
chloroform (250 mL) was added. The resulting slurry was filtered
through sand and concentrated under vacuum to give an orange solid
(5.2 g, 43%).
Example 1(ii)
SYNTHESIS OF COMPOUND 2
[0044] Compound 1 (4 g, 11.2 mmol) was dissolved in methanol ((150
mL) and was cooled in an ice/water bath. Sodium borohydride (8 g,
208 mmol) was added portion wise over 30 minutes. The mixture was
left to rise to room temperature with stiffing over 16 hours. The
solution was concentrated to dryness under vacuum to give an off
white solid. The solid was dissolved in water (100 mL) and was
heated to 60.degree. C. for half an hour during which time an oily
material formed in the mixture. THF (100 mL) was added and the
organic layer was separated. The aqueous layer was extracted again
with THF (100 mL). The combined extracts were filtered through a
phase separator cartridge and were concentrated to dryness under
vacuum. The oily solids were re-dissolved in THF (20 mL) and water
(15 mL) was added. The solution was concentrated slowly until a
white solid crystallized which was collected by filtration, washed
with ice cold water and dried under high vacuum (1.6 g, 38%).
Example 1(iii)
Synthesis of Compound 3
[0045] Compound 2 (0.1 g, 0.270 mmol) was dissolved in dry DMF (5
mL) and potassium carbonate added (1.1 eq. 0.297 mmol, 0.041 g).
The alkyl bromide (1.1 eq. 0.297 mmol, 81.7 mg) was added portion
wise following the reaction by HPLC-mass spectrometry by taking
approximately 0.1 mL volume from the reaction and diluting with 1:1
0.1% formic acid in water:acetonitrile (10 mL). The reaction was
stirred at room temperature for 16 hours. A further 0.25
equivalents of the alkyl bromide was added and the reaction stirred
for a further 16 hours. The reaction mixture was concentrated to
dryness under vacuum. This was used in the next step without
further purification.
Example 1(iv)
Synthesis of Compound 4
[0046] Crude compound 3 was dissolved in dry DMF (20 mL) and
pyridine (2 mL) was added followed by di-tert-butylcarbonate (1 g,
4.58 mmol, 17 eq.). The mixture was heated at 70 .degree. C. under
nitrogen for 16 hours. The crude product was analysed by thin layer
chromatography (silica gel plates eluting with 10% methanol/DCM)
and by LCMS. Thin layer chromatography showed two major spots
having Rf values of 0.2 and 0.5 and some minor spots. The mixture
was purified by flash column chromatography on silica gel eluting
with 100% petrol 40-60 to 100% ethyl acetate. The second major peak
was shown to be the desired penta-BOC product by NMR and LCMS (50
mg).
Example 2
##STR00029##
[0047] Example 2(i)
Synthesis of Compound 5
[0048] Compound 2 (0.1 g, 0.270 mmol) was dissolved in dry DMF (2
mL) and a solution of the alkyl bromide (1.1 eq. 0.297 mmol, 81.07
mg) in dry DMF (1 mL) was added over 5 minutes. The solution was
stirred at room temperature for 16 hours. The DMF was removed under
reduced pressure and white solids dissolved in an minimum volume of
water/methanol (1:1). Preparative HPLC (Phenomenex luna C18(2)
150.times.21.2, acetonitrile/water 5% to 70% over 10 minutes) gave
a major peak having t.sub.r of 8-8.5 minutes which was freeze dried
giving an white solid (15 mg). NMR and LCMS confirmed the
structure.
Example 2(ii)
Fluoride Binding Studies with [.sup.19F]-fluoride
[0049] Compound 5 (1 mg) in water (0.1 mL) acidified to pH 1 with
1N HCl and an aqueous solution of potassium fluoride (0.1-1 eq) was
added at RT. The solutions were analysed by reversed phase HPLC (1%
TFA/water, 1% TFA MeCN gradient on Luna C5 150.times.4.6 mm,
detecting at 254 nm).
Example 2(iii)
Fluoride Radiolabelling of Compound 5 with [.sup.18F]-fluoride
##STR00030##
[0051] 1M HCl (4.54, 4.5 mmol) was added to compound 5 (0.1 mg, 180
nmol) in 50:50 methanol/water (0.2 mL). This acidified solution was
added directly to a glass vial containing [.sup.18F]fluoride (98
MBq) in target water (0.05 mL) and left at room temperature for 20
minutes. The reaction was analayzed by reverse phase HPLC (solvent
A=0.1% TFA in water; Solvent B=0.1% TFA in MeCN, Luna C5
150.times.4.6 mm, detecting at 254 nm; Gradient: 0 to 3 minutes (2%
B), 3-10 minutes (2 to 70% B), 10 to 13 minutes (70% B); 13 to 16
minutes (70 to 2% B), 16 to 21 minutes (2% B); flow rate 1
mL/minute. [.sup.18F]-5 has a retention time of 10.1 minutes.
[.sup.18F]-5 was purified using the same HPLC method with a decay
corrected isolated yield of 64%. Purified [.sup.18F]-5 is stable
(>95% RCP) in an acidic solution (pH<3).
[0052] Increasing the pH [.sup.18F]-5 solution to pH 7 results in
the removal of more than 70% of the [.sup.18F]-fluoride from the
cryptand as measured by HPLC peak intensity.
[0053] HPLC Conditions:
0-3 mins 2% (B) 3-10 mins 2-70% (B) 10-13 mins 70% (B) 13-16 mins
70-2% (B) 16-21 mins 2% (B)
Column Luna C5 150.times.4.6 mm
[0054] Eluent Solvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in
acetonitrile Pump speed 1 mL/min,
Wavelength 254 nm
[0055] Example 3-synthesis of a resin bead-bound solid supported
cryptand
##STR00031##
[0056] Compound 2 and a base e.g. potassium carbonate are dissolved
in dry DMF (2 mL) and is added to a suspension of
(chloromethyl)polystyrene resin (e.g. Merrifield's peptide resin
available from Sigma-Aldrich) in dry DMF (1 mL) was added over 5
minutes. The mixture is agitated at room temperature or elevated
temperature until the free cryptand starting material is consumed
(determined by LCMS). The DMF was removed by filtration and the
resin washed a number of times with one or more organic solvents
e.g. methanol, dichloromethane or dimethylformamide. The final
resin is characterized by elemental analysis.
Example 2
Synthesis of a Resin Bead-Bound Solid Supported Cryptand
##STR00032##
[0058] Compound 5 is treated with an excess of tBOC anhydride to
give the corresponding penta-BOC protected species. Saponification
of the methyl ester to give the free acid, followed by amide bond
formation with an amino-functionalized resin (e.g.
(aminomethyl)polystyrene resin available from Sigma-Aldrich) using
a coupling reagent (e.g. HATU) gives the solid-supported BOC
protected cryptand. Finally, BOC de-protection using
trifluofoacetic acid gives the desired solid-supported cryptand.
This is characterized by NMR and elemental analysis.
* * * * *